Abstract: Tau and amyloid-? immunotherapies are the most common approaches by the pharmaceutical industry to tackle Alzheimer's disease, which is characterized by such depositions. The majority of these promising therapies are whole monoclonal antibodies (mAbs). Much less attention has been paid to antibody fragments, like single-chain variable fragments (scFvs) and single domain antibodies (sdAbs), which have certain advantages that justify exploring their therapeutic and diagnostic potential. Of the two targets, tau may be more promising because its pathology correlates better with dementia than A? lesions. The aims of the proposal are: 1) To clarify with in vivo two-photon imaging approaches the kinetics, mechanisms, and functional consequences of tau antibody therapies, including their fragments, and; 2) To determine the diagnostic imaging potential of the smallest biological binders, sdAbs, to the tau protein. Based on our preliminary data, it is hypothesized that in vivo two-photon imaging is the most sensitive and comprehensive in vivo approach, within a short timeframe, to clarify kinetics and therapeutic potential of tau mAbs/scFvs/sdAbs. Furthermore, this technique should provide a mechanistic insight into how the therapies work, and benefit neuronal function. Likewise, it is hypothesized that the small size of the sdAbs will provide diagnostic benefits over the larger mAbs and scFvs, primarily because of greater access into the brain and to the tau target, and to some extent due to their binding to novel epitopes. We have applied and further developed various two-photon imaging approaches to clarify the mechanisms of tau antibody therapies. Specifically, co-injection of a brain permeable tau ?-sheet dye, FSB, with a fluorescently labeled tau mAb/scFv/sdAb, allows for examining the kinetics of their colocalization with tau aggregates and subsequent clearance of tau lesions. Furthermore, viral neuronal expression of calcium and glutamate indicators provides a window into functional deficits associated with tau pathology, and allow study of the ability of tau mAbs/scFvs/sdAbs to attenuate and/or reverse these signaling abnormalities. For these studies, we have 45 clones of tau mAbs, numerous scFvs, and 50 clones of sdAbs with unique binding regions that recognize various forms of the tau protein. For the therapeutic and mechanistic studies, we will examine a few of these that based on our published and preliminary data have shown potential to clear pathological tau and prevent its toxicity. For the diagnostic studies, up to 20 sdAbs will be selected based on various in vitro binding assays for in vivo imaging studies using the In Vivo Imaging System (IVIS). Subsequently, the most promising ones will be examined further as potential diagnostic positron emission tomography (PET) ligands by imaging tauopathy mice in a microPET. This proposal, with strong relevance to other protein aggregation diseases, should clarify the mechanisms of tau immunotherapies and may provide therapeutic and diagnostic candidates for clinical trials.